JP6507791B2 - Nucleic acid detection method - Google Patents

Description

  The present invention relates to a method for conveniently and sensitively detecting a target nucleic acid contained in a sample, and a kit using the method.

  In gene diagnosis where diagnosis is performed based on the presence or absence or amount of target nucleic acid contained in a sample, the amount of the target nucleic acid is extremely small and sufficient detection sensitivity can not be obtained as it is. The target nucleic acid is amplified using a nucleic acid amplification method such as the method (Patent Documents 1 and 2), the TMA method (Patent Document 3), and the TRC method (Patent Document 4 and Non-patent Document 1).

  As a method for detecting the amplified target nucleic acid, conventionally, electrophoresis using an agarose gel or the like, or nucleic acid hybridization using a labeled oligonucleotide probe capable of hybridizing with a part of the target nucleic acid under stringent conditions Hybridization methods are known. However, since the electrophoresis method is carried out by a manual method and the reagent (intercalator) used for target nucleic acid staining contains a reagent having carcinogenicity, pay sufficient attention to the handling and disposal of the reagent. There is a need. The nucleic acid hybridization method can automatically detect the target nucleic acid by adding the previously labeled probe to the amplification reagent, so that the risk relating to the handling and disposal can be reduced. However, detection using nucleic acid hybridization requires a special fluorescent substance and a dedicated detection device.

  As a simpler method of detecting amplified nucleic acid, a nucleic acid chromatography method of detecting the target nucleic acid by adding a solution or reagent containing the amplified target nucleic acid to a porous membrane and developing the same by capillary action (Patent Documents 5 and 6). The nucleic acid chromatography method is a method that does not require a dedicated detection device or the like, and thus is particularly excellent as a clinical genetic test method. However, in the conventional nucleic acid chromatography method, a step of amplifying a target nucleic acid in a sample, a step of binding a reagent (chromogenic molecule) that develops color upon binding to the target nucleic acid and the target nucleic acid, and bound to the chromogenic molecule The problem is that it requires three steps of detecting the target nucleic acid, and the operation is complicated.

Patent No. 2650159 Patent No. 3152927 Patent No. 3241717 Japanese Patent Laid-Open No. 2000-014400 Japanese Patent Application Publication No. 2001-157598 Patent 4268944 gazette

Ishiguro, T .; et al, Analytical Biochemistry, 314, 77-86 (2003).

  An object of the present invention is to provide a method for simply and sensitively detecting a target nucleic acid contained in a sample without using a dedicated detection device, and a kit using the method.

  As a result of intensive studies to solve the above problems, the present inventors have completed the present invention.

That is, the first aspect of the present invention is a method for detecting a target nucleic acid contained in a sample, which comprises the following steps (1) to (3).
(1) amplification of a target nucleic acid contained in a sample, and specific binding of the amplified target nucleic acid and a first binding agent bound to a labeling substance simultaneously obtained in the step (2) (1) In step (3) (2), in which the complex of the target nucleic acid and the first binding factor and the second binding factor bound to the membrane are specifically bound by chromatography using a developing solution Detecting the complex of the obtained target nucleic acid, the first binding factor and the second binding factor with the labeled substance bound to the first binding factor In the second aspect of the present invention, the labeled substance is visible The detection method according to the first aspect is a substance that absorbs or reflects light.

  In the third aspect of the present invention, the first or second binding agent is an oligonucleotide comprising a base sequence capable of hybridizing to a target nucleic acid under stringent conditions. The detection method according to the aspect of

  In the fourth aspect of the present invention, the target nucleic acid amplified in the step (1) has a plurality of types, and a plurality of types of the first binding agent and / or the second binding agent exist. It is a detection method according to any of the three aspects.

  The fifth aspect of the present invention is the detection method according to any one of the first to the fourth, wherein the developing solution at least contains a nonionic surfactant.

  A sixth aspect of the present invention is the detection method according to any one of the first to fifth aspects, wherein amplification of the target nucleic acid is performed by any one of NASBA method, TMA method and TRC method.

Furthermore, the seventh aspect of the present invention is
An enzyme and a primer for amplifying a target nucleic acid contained in a sample, an oligonucleotide containing a base sequence capable of hybridizing under stringent conditions with the target nucleic acid to which a labeling substance is bound, and a non-ionic surfactant A reagent containing
A membrane bound to an oligonucleotide containing a base sequence capable of hybridizing to the target nucleic acid under stringent conditions;
And a kit for detecting a target nucleic acid.

  The eighth aspect of the present invention is the kit according to the seventh aspect, comprising a plurality of oligonucleotides containing a nucleotide sequence capable of hybridizing under stringent conditions with a target nucleic acid to which a labeling substance is bound. .

  The ninth aspect of the present invention is the membrane according to the seventh or eighth aspect, wherein the membrane is a membrane in which a plurality of types of oligonucleotides comprising a base sequence capable of hybridizing to a target nucleic acid under stringent conditions are bound. Detection kit.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the target nucleic acid is a region of single-stranded or double-stranded DNA or RNA derived from at least one cell, bacteria, fungus, virus, etc., which is amplified in the above step (1). Say When the target nucleic acid is single-stranded DNA or RNA, their complementary strands are also included in the target nucleic acid. When nucleic acid amplification using the TRC method is performed in the step (1), the length of the target nucleic acid is preferably 100 to 300 bases, and particularly preferably 100 to 200 bases.

  When detecting a target nucleic acid contained in a sample using the detection method of the present invention, it is preferable to carry out a step of extracting the target nucleic acid from the sample in advance. The method for extracting the target nucleic acid is not limited, and the EDTA-SDS-phenol-ethanol method, lithium chloride-urea method, protease K-deoxyribonuclease method, phenol-SDS method, guanidine thiocyanate-cesium chloride method, guanidine thiocyanate-trifluoro method Cesium acetate method, acidic guanidine thiocyanate-phenol-chloroform method (AGPC method), vanadyl ribonucleoside composite method, magnetic silica method can be exemplified. Alternatively, a commercially available nucleic acid extraction kit may be used.

  As a nucleic acid amplification method used at the process of said (1), well-known methods, such as PCR method, NASBA method, TMA method, and TRC method, can be used. Furthermore, for detection of the amplified target nucleic acid, an oligonucleotide (a first binding agent) comprising a base sequence capable of hybridizing with the target nucleic acid bound with a labeling substance under stringent conditions, and a condition stringent to the target nucleic acid When using a membrane bound with an oligonucleotide (second binding factor) containing a base sequence capable of hybridizing in a single strand, asymmetric PCR that can amplify single-stranded DNA or RNA, NASBA method, TMA method, TRC method In particular, NASBA method, TMA method and TRC method, which can amplify single stranded nucleic acid at a constant temperature, are preferable. In the present specification, stringent conditions can be selected from known conditions and are not particularly limited. For example, at 42 ° C., 50% (v / v) formamide, 0.1% bovine serum Conditions that can be hybridized under conditions in which albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate coexist The conditions which can be hybridized under the nucleic acid amplification conditions described in the Examples of the specification can be mentioned.

  In the present invention, the labeling substance to be bound to the first binding factor is the binding of the second binding factor bound to the membrane, the first binding factor and the target nucleic acid, and the first binding factor is constant on the membrane Any substance can be used as long as it can be detected by collecting the amount, for example, colored polymer particles, gold colloids, dyes, and fluorescent substances. In particular, when a substance that absorbs or reflects visible light is used as a labeling substance, it is preferable to collect a predetermined amount of the first binding agent, which enables visual detection. The binding between the first binding agent and the labeling substance can be appropriately selected depending on the aspect of the binding agent, for example, covalent binding, electrostatic binding, antigen-antibody binding, ligand-receptor binding, biotin-avidin binding, internucleotide And combinations thereof.

  In the present invention, the first binding agent and the second binding agent may be substances capable of specifically binding to the amplified target nucleic acid, but a base sequence capable of hybridizing with the target nucleic acid under stringent conditions It is preferable to use an oligonucleotide containing the compound as a first binding agent and a second binding agent. The length of the oligonucleotide in the preferred example may be 8 bases or more, preferably 15 bases or more, and more preferably 18 bases or more. The oligonucleotide may be DNA, RNA, or a chimera thereof, and may further have artificial bases or chemical modifications. When the target nucleic acid amplified in the step (1) is modified, a substance corresponding to the modified substance can also be used as a first binding factor and a second binding factor. For example, positive charge-negative charge, antigen-antibody, ligand-receptor, biotin-avidin, or a combination thereof can be mentioned.

The membrane to which the second binding agent is immobilized in the present invention is
A substance capable of binding directly or indirectly to the second binding agent,
A substance capable of separating the complex from the solution containing the complex of the target nucleic acid and the first binding factor obtained in the step (1) by chromatography using a developing solution,
And a substance capable of accumulating the labeled substance bound to the first binding factor when the complex and the second binding factor are bound,
There is no limitation as long as it has a porous membrane such as nitrocellulose or nylon. Furthermore, in order to strengthen the bond with the second binding agent, the solid phase may be subjected to suitable processing or introduction of a functional group or a binding agent. The binding between the second binding agent and the solid phase can be appropriately selected according to the aspect of the binding agent, for example, covalent binding, electrostatic binding, antigen-antibody binding, ligand-receptor binding, biotin-avidin binding, internucleotides Hybridization and combinations thereof can be mentioned.

  In the present invention, chromatography is a method of moving and developing a solution in a certain direction by capillary action, and thin film chromatography, paper chromatography, capillary chromatography and the like can be exemplified. Moreover, as a shape, the thing of the kit type | mold thing and the dipstick type | mold which have a water-absorptive substrate for expansion | deployment, an expansion liquid, and an expansion layer can be illustrated.

  In the detection method of the present invention, when there are a plurality of types of target nucleic acids to be detected, the amplification of the plurality of types of target nucleic acids in the step (1) and one type (or plurality of types) The specific binding with the first binding factor is carried out, and in the step (2), the complex of the target nucleic acid and the first binding factor with one or more types of second binding bound to the membrane Specific binding with an agent may be performed. As an example of a plurality of types of first binding agents, an embodiment in which a labeling substance is bound to an oligonucleotide containing a base sequence capable of hybridizing under stringent conditions to each target nucleic acid to be detected can be mentioned. The labeling substance may be a common labeling substance or may be a labeling substance having different properties (such as color). As an example of a plurality of types of second binding agents, an embodiment in which an oligonucleotide containing a base sequence capable of hybridizing under stringent conditions to each target nucleic acid to be detected is bound to a membrane can be mentioned.

  In the detection method of the present invention, in the step (2), the complex with the target nucleic acid and the first binding factor is specifically bound to the second binding factor by chromatography using a developing solution. However, the developing solution used herein may contain a nonionic surfactant. While the separation by chromatography is promoted by adding the nonionic surfactant to the developing solution, the nonionic surfactant does not inhibit the amplification reaction of the target nucleic acid performed in the step (1). . In addition, the reaction solution of the step (1) (for example, an enzyme and a primer for amplifying a target nucleic acid contained in a sample, and a base capable of hybridizing with the target nucleic acid bound with a labeling substance under stringent conditions It is preferable to previously add a nonionic surfactant to a solution containing an oligonucleotide primer containing a sequence, since the reaction solution after the step (1) can be used as it is as a developing solution. The nonionic surfactant can be appropriately selected from nonionic surfactants generally used by those skilled in the art, and Tween 20, Tween 60, Tween 80, Triton X-100 (all trade names) It can be illustrated. Among them, Tween 20 is preferred as the nonionic surfactant added to the developing solution. Furthermore, if the membrane is previously dipped in a solution containing a nonionic surfactant before the step (2), the detection step of the above (3) is easier (eg, sharper visual detection is possible). It is preferable because

  The method for detecting a target nucleic acid of the present invention comprises a first step of simultaneously amplifying target nucleic acid contained in a sample and specifically binding the amplified target nucleic acid to a first binding agent bound to a labeling substance. Second step of specifically binding the complex of the target nucleic acid and the first binding agent obtained in the first step to the second binding agent bound to the membrane by chromatography using a developing solution And a third step of detecting the complex of the target nucleic acid, the first binding agent and the second binding agent obtained in the second step with a labeled substance bound to the first binding agent. It is characterized by

  By the method of the present invention and the kit of the present invention using the above method, the target nucleic acid contained in the sample can be detected conveniently and with high sensitivity. When a substance that absorbs or reflects visible light is used as the labeling substance bound to the first binding factor, the presence or absence of the target nucleic acid can be detected visually, and there is no need to use a special device. Time and the number of steps can be reduced.

Structure of the intercalator fluorescent dye-labeled nucleic acid probe. B ¹ , B ² , B ³ and B ^{4 each} represent a base. In addition, in order to prevent the extension reaction from 3 'terminal side -OH, glycolic acid modification is made to 3' terminal side -OH. The figure which showed the result of having detected influenza virus RNA using the detection method of this invention. The figure which showed the result of having detected each type of influenza virus RNA using the detection method of this invention.

  Hereinafter, the present invention will be described in more detail using an example in which influenza virus RNA and RegIV are used as target nucleic acids, but the present invention is not limited to this example.

Example 1 Preparation of Influenza Virus Standard RNA Influenza virus RNA (hereinafter referred to as standard RNA) used in the examples described later was prepared by the method shown below.
(1) SEQ ID NO: 15 (H1N1 subtype A influenza virus, segment 5, cDNA partial sequence, nucleotide sequence from 410th to 930th nucleotide sequence of GenBank No. FJ 969536), SEQ ID NO: 16 (H3N2 subtype A influenza virus, Segment 5, cDNA partial sequence, base sequence from 455th to 975th in GenBank No. NC — 007369 (where C is T in 663) and SEQ ID NO: 17 (Influenza B virus, segment 7, cDNA partial sequence, GenBank No A double-stranded DNA consisting of the nucleotide sequence described in nucleotides 206 to 735 of CY115184 was prepared and inserted into T-Vector pMD20 (Takara Bio Inc.) (complementary strand of the cDNA sequence) The SP6 promoter is added to the 5 'end of
(2) The plasmid DNA prepared in (1) was digested with restriction enzyme at the 5 'end of the inserted influenza virus cDNA sequence to prepare a linear DNA.
(3) In vitro transcription by SP6 RNA polymerase was performed using the DNA prepared in (2) as a template. Thereafter, the template DNA was completely digested by DNase I treatment and purified to prepare a standard RNA. The prepared RNA was quantified by measuring the absorbance at 260 nm.

  The total length of the standard RNA prepared in this example is about 500 bases, which is a part of the full-length influenza virus RNA (segment 5: about 1500 bases, segment 7: about 1100 bases). Is sufficiently applicable.

Example 2 Preparation of first binding agent bound to labeling substance Latex particles 500 μm in diameter Estapor (manufactured by Merck) are used as the labeling substance, and the labeling substance is made stringent to influenza virus RNA by the method described below. It was bound to a hybridizable oligonucleotide (first binding agent) under conditions.
(1) 150 ng of each of oligonucleotides containing the sequence described in SEQ ID NOS: 10 to 12 modified at the 3 'end with an amino group is added to 1.4 × 10 ¹⁰ latex particles, and left at room temperature for 30 minutes did. SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 are base sequences capable of hybridizing under stringent conditions.
(2) Further, water-soluble carbodiimide was added to a final concentration of 10 mg / mL, and allowed to react at room temperature for 2 hours to bind latex particles to the oligonucleotide.
(3) The precipitate obtained by centrifugation (the first binding agent bound to the labeling substance) is washed with a solution containing Tween 20 and SDS, and buffer (0.5 mM EDTA, 1 M NaCl, and 0.05% Tween) It was stored in 10 mM Tris-HCl (pH 7.4) containing 20.

Example 3 Preparation of Second Binding Factor Bound to Membrane As a membrane, a nitrocellulose membrane HF120 (manufactured by Merck) bonded to a backing sheet (manufactured by Merck) is used, and influenza virus is added to the membrane by the method described below. A hybridizable oligonucleotide (second binding agent) was bound under stringent conditions with RNA.
(1) 50 μM of an oligonucleotide consisting of the sequence of SEQ ID NO: 13 and / or 14 was applied to a membrane using a coater. Note that SEQ ID NO: 13 and SEQ ID NO: 14 are base sequences that can hybridize with SEQ ID NO: 17 under stringent conditions.
(2) After irradiating UV for 2 minutes, the oligonucleotide was attached to the membrane by drying at 37 ° C. for 2 hours.

Example 4 Preparation of Nucleic Acid Probe Labeled with Intercalator Fluorescent Dye According to the method of Ishiguro et al. (Ishiguro, T. et al, Nucleic Acids Res., 24, 4992-4997 (1996)). A linker is attached to the phosphate diester moiety between the 17th T and the 18th T from the 5 'end of the sequence, and the 8th A to the 9th T from the 5' end of the sequence described in SEQ ID NO: 8 A nucleic acid probe (hereinafter referred to as an INAF probe) labeled with an intercalating fluorescent dye was prepared by binding oxazole yellow through it (FIG. 1). SEQ ID NO: 7 and SEQ ID NO: 8 are base sequences that can hybridize with SEQ ID NO: 17 under stringent conditions.

Example 5 Examination of Developing Solution Surface activity of the reaction solution used in the step of simultaneously performing amplification of the target nucleic acid contained in the sample and specific binding of the amplified target nucleic acid and the first binding factor bound to the labeling substance. It was examined whether chromatography could be achieved by adding an agent.
(1) The prepared RegIV standard RNA (SEQ ID NO: 22) was diluted to 10 ³ copies / 5 μl with TE buffer and used as an RNA sample.
(2) After dispensing 17 μL of the reaction solution having the following composition into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), the oligonucleotide described in SEQ ID NO: 20 prepared in Example 2 was bound 10 ⁹ (2 μL) of latex particles were added, and an additional 3 μL of different concentrations and types of surfactant were added.

Composition of the reaction solution: final concentration after addition of the starting solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
Each 0.25 mM dATP, dCTP, dGTP, dTTP
2.6mM ATP, CTP, GTP, TTP each
3.06mM ITP
70 mM trehalose 0.2 μM first primer (SEQ ID NO: 18): The T7 promoter sequence (SEQ ID NO: 9) is added to the 5 'end of the base sequence described in each SEQ ID NO: Second primer (SEQ ID NO: 19)
6.4 U AMV Reverse Transcriptase (Life Science)
142 U T7 RNA polymerase (3) The above reaction solution was kept at 46 ° C. for 4 minutes, and then 8 μL of the starting solution having the following composition was added.

Composition of starting solution: final concentration after addition of starting solution (in 30 μL) 21.2 mM magnesium chloride 102.7 mM potassium chloride 1.2% glycerol 11.5% DMSO
(4) In the above mixed solution, the membrane bound with the oligonucleotide having the sequence of SEQ ID NO: 21 prepared in Example 3 was immersed and subjected to chromatography.

  The results are shown in Table 1. It can be seen that the addition of a non-ionic surfactant enables chromatography.

実施例６ 界面活性剤の添加による核酸増幅反応への影響
試料中に含まれる標的核酸の増幅および前記増幅した標的核酸と標識物質を結合した第１の結合因子との特異的結合を同時に行なう工程で用いる反応液に、界面活性剤を添加することで前記標的核酸の増幅反応に影響をおよぼすか検討した。
（１）実施例１で調製したＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルス標準ＲＮＡ（配列番号１５）を、ＴＥバッファーを用いて１０^３コピー／５μＬとなるように希釈し、これをＲＮＡ試料として用いた。
（２）以下の組成の反応液１４μＬを０．５ｍＬ容量ＰＣＲチューブ（Ｉｎｄｉｖｉｄｕａｌ Ｄｏｍｅ Ｃａｐ ＰＣＲ Ｔｕｂｅ、ＳＳＩ製）に分注し、これに前記ＲＮＡ試料５μＬを添加した。さらに実施例２で作製した配列番号１０に記載のオリゴヌクレオチドを結合したラテックス粒子１０^９個（２μＬ）と、種々の濃度および種類の界面活性剤溶液３μＬを添加した。

Example 6 Influence on nucleic acid amplification reaction by addition of surfactant Amplification of target nucleic acid contained in a sample and step of simultaneously performing specific binding of the amplified target nucleic acid and the first binding agent bound to a labeling substance It was examined whether addition of a surfactant to the reaction solution used in No. 1 affects the amplification reaction of the target nucleic acid.
(1) The type A (H1N1 subtype) influenza virus standard RNA (SEQ ID NO: 15) prepared in Example 1 is diluted with TE buffer to 10 ³ copies / 5 μL, and this is used as an RNA sample It was.
(2) 14 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of the RNA sample was added thereto. Furthermore, 10 ⁹ particles (2 μL) of latex particles conjugated with the oligonucleotide shown in SEQ ID NO: 10 prepared in Example 2 and 3 μL of surfactant solutions of various concentrations and types were added.

Composition of the reaction solution: final concentration after addition of the starting solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
Each 0.25 mM dATP, dCTP, dGTP, dTTP
2.6mM ATP, CTP, GTP, TTP each
3.06mM ITP
70 mM Trehalose 20 nM INAF probe (SEQ ID NO: 7) (prepared in Example 4)
0.2 μM first primer (SEQ ID NO: 1): A T7 promoter sequence (SEQ ID NO: 9) is added to the 5 'end of the base sequence described in each SEQ ID NO: Primer (SEQ ID NO: 2)
6.4 U AMV Reverse Transcriptase (Life Science)
(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 8 μL of an initiator solution having the following composition was added.

Starting solution composition: final concentration after addition of initiator (in 30 μL) 21.2 mM magnesium chloride 102.7 mM potassium chloride 1.2% glycerol 11.5% DMSO
(4) Subsequently, using a fluorescence spectrophotometer (TRCRapid-160, manufactured by Tosoh Corporation) capable of directly measuring a PCR tube, the reaction is carried out at 46 ° C. and at the same time the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (value obtained by dividing the fluorescence intensity value for a predetermined period of time by the background fluorescence intensity ratio) exceeded 1.2, with the addition of the initiator solution as 0 minutes.

  The results are shown in Table 2. It can be seen that the Tween (trade name) system among nonionic surfactants does not affect the amplification reaction of the target nucleic acid even at a relatively high concentration (about 1% (w / v)). On the other hand, since Triton X-100 (trade name) inhibits the amplification reaction of the target nucleic acid at 0.05% (w / v), Triton X-100 (trade name) is used as a nonionic surfactant. When used, it can be seen that it is necessary to add at relatively low concentrations (less than 0.05% (w / v)).

Example 7 Visual Detection of Influenza Virus RNA (Part 1)
(1) The surfactant added in Example 6 (2) is 0.05% (w / v) Tween 20 (trade name), and the reaction time of Example 6 (4) is 10 minutes. The same operation as in Example 6 was performed.
(2) The membrane to which the oligonucleotide consisting of the sequence of SEQ ID NO: 13 prepared in Example 3 was bound was placed in a PCR tube containing the reaction solution after the operation of (1), and chromatography was performed.

The results are shown in FIG. About 1 minute after the start of chromatography, it was possible to confirm the band derived from the amplified influenza virus RNA.

Example 8 Visual Detection of Influenza Virus RNA (Part 2)
(1) Type A (H1N1 subtype) influenza virus standard RNA (SEQ ID NO: 15) prepared in Example 1, type A (H3N2 subtype) influenza virus standard RNA (SEQ ID NO: 16) and type B influenza virus standard RNA ( SEQ ID NO: 17) was diluted to 10 ³ copies / 5 μl each with TE buffer, and these were used as RNA samples.
(2) 14 μL of the reaction solution having the following composition was dispensed into 0.5 mL volume PCR tubes (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of any of the RNA samples was added thereto. Further, 10 ⁹ pieces (2 μL) of latex particles to which oligonucleotides described in SEQ ID NOs: 10 to 12 prepared in Example 2 were attached and 2 μl of Tween 20 (trade name) at various concentrations were added.

Composition of the reaction solution: final concentration after addition of the starting solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
Each 0.25 mM dATP, dCTP, dGTP, dTTP
2.6mM ATP, CTP, GTP, TTP each
3.06mM ITP
70 mM Trehalose 20 nM INAF probe (SEQ ID NOS: 7 and 8) (prepared in Example 4)
Each 0.2 μM first primer (SEQ ID NOs: 1, 3 and 5): T7 promoter sequence (SEQ ID NO: 9) is added to the 5 'end of the base sequence described in each SEQ ID NO: 0.2 μM each second primer (SEQ ID NO: 2, 4 and 6)
6.4 U AMV Reverse Transcriptase (Life Science)
(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 8 μL of an initiator solution having the following composition was added.

Starting solution composition: final concentration after addition of initiator (in 30 μL) 21.2 mM magnesium chloride 102.7 mM potassium chloride 1.2% glycerol 11.5% DMSO
(4) Subsequently, using a fluorescence spectrophotometer (TRCRapid-160, manufactured by Tosoh Corporation) capable of directly measuring a PCR tube, the reaction is carried out at 46 ° C. and at the same time the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (value obtained by dividing the fluorescence intensity value for a predetermined period of time by the background fluorescence intensity ratio) exceeded 1.2, with the addition of the initiator solution as 0 minutes.
(5) In the solution after the reaction of (4), the membrane to which the oligonucleotide consisting of the sequence of SEQ ID NO: 13 and 14 prepared in Example 3 was attached was placed, and chromatography was performed.

Be added latex particles bound oligonucleotides by each 10 ^9, by adding Tween 20 (trade name), it was possible to suppress the influence on the amplification reaction of the target nucleic acid (Table 3). Moreover, as a result of performing chromatography about the reaction liquid after adding 0.05% (w / v) Tween 20 (brand name) and performing amplification reaction, each influenza virus amplified about 1 minute after the start A band derived from RNA could be identified (FIG. 3).

Claims (6)

A method for detecting a target nucleic acid contained in a sample, comprising the following steps (1) to (3) :
The target nucleic acid is RNA,
The nonionic surfactant is 0.01 to 5% (w / v) Tween 20, 0.01 to 10% (w / v) Tween 60, 0.01 to 10% (w / v) v) Tween 80, and any selected from 0.01 to less than 0.05% (w / v) Triton X-100:
(1) amplification of a target nucleic acid contained in a sample and specific binding between the amplified target nucleic acid and a first binding agent bound to a labeling substance in the same reaction solution ,
The reaction solution contains a target nucleic acid, a first binding agent to which a labeling substance is bound, a nonionic surfactant, a reverse transcriptase, a first primer, a second primer and an RNA polymerase, and the first primer and At least one of the second primers has a promoter sequence;
(2) The complex of the target nucleic acid obtained in the step of (1) and the first binding factor, and the second binding factor bound to the membrane were used as a reaction solution after the reaction of (1) Specifically binding by chromatography ;
(3) detecting the complex of the target nucleic acid, the first binding agent and the second binding agent obtained in the step of (2) with the labeling substance bound to the first binding agent . The detection method according to claim 1, wherein the labeling substance is a substance that absorbs or reflects visible light. The detection method according to claim 1 or 2, wherein the first binding agent and the second binding agent are oligonucleotides comprising a base sequence capable of hybridizing to a target nucleic acid under stringent conditions. The detection method according to any one of claims 1 to 3, wherein there are a plurality of types of target nucleic acids amplified in the step (1), and a plurality of types of first binding agents and / or second binding agents are present. The detection method according to any one of claims 1 to 4 , wherein amplification of the target nucleic acid is performed by any one of NASBA method, TMA method and TRC method. The first binding agent to which the labeling substance is bound is a particle to which an oligonucleotide is bound, and the number of said particles in the reaction solution is 5 x 10 ⁸ ~ 10 ⁹ The detection method according to any one of claims 1 to 5, wherein

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